Ultrastructural and molecular confirmation of the development of Sarcocystis neurona tissue cysts in the central nervous system of southern sea otters (Enhydra lutris nereis)

Abstract

In 2004, three wild sea otters were diagnosed with putative Sarcocystis neurona-associated meningoencephalitis by histopathology and immunohistochemistry. Schizonts, free merozoites and tissue cysts were observed in the brains of all three infected animals. Tissue cysts from sea otter 1 (SO1) stained positively using anti-S. neurona polyclonal antiserum. However, positive staining does not preclude infection by closely related or cross-reactive tissue cyst-forming coccidian parasites. Two immature tissue cysts in the brain of SO1 were examined using transmission electron microscopy. Ultrastructural features included cyst walls with thin villous projections up to 1 μm long with tapered ends and a distinctive, electron-dense outer lining layer composed of linearly-arranged, semi-circular structures with a "hobnailed" surface contour. Small numbers of microtubules extended down through the villi into the underlying granular layer. Metrocytes were short and plump with an anterior apical complex, 22 subpellicular microtubules, numerous free ribosomes and no rhoptries. Some metrocytes appeared to be dividing, with two adjacent nuclear profiles. Collectively these ultrastructural features were compatible with developing protozoal cysts and were similar to prior descriptions of S. neurona tissue cysts. Panspecific 18S rDNA primers were utilized to identify protozoa infecting the brains of these otters and DNA amplification and additional sequencing at the ITS1 locus confirmed that all three otters were infected with S. neurona. No other Sarcocystis spp. were detected in the brains or skeletal muscles of these animals by immunohistochemistry or PCR.*Corresponding authors. Tel.: +1 831 469 1746; Fax: +1 831 469 1723, E-mail address: mmiller@ospr.dfg.ca.gov. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. We believe this is the first ultrastructural and molecular confirmation of the development of S. neurona tissue cysts in the CNS of any animal. NIH Public Access

An update on Sarcocystis neurona infections in animals and equine protozoal myeloencephalitis (EPM)

“…Sarcocysts in brain are often round, and smaller in size than in skeletal muscle (Thomas et al, 2007; Miller et al, 2009). Additionally, sarcocysts in myocardium are smaller than those in skeletal muscle.…”

“…Primers developed within the ITS1 (called ITS1 500 ) are now routinely used to identify infection by opossum derived Sarcocystis spp. and DNA sequencing of the PCR amplicons distinguish between S. speeri, S. lindsayi, S. falcatula, and S. neurona (Miller et al, 2009). …”

“…Most of these deaths were sporadic. An epizootic of protozoal encephalitis in sea otters was recorded by Miller et al (2009, 2010). Within one month, 63 sick and dead otters were recovered near Morro Bay, California.…”

“…Microscopically, meningoencephalitis in association with schizonts and myositis associated with sarcocysts predominated. Immature and mature sarcocysts were seen in muscles, heart, and brain (Miller et al, 2009; Miller et al, 2010). …”

The mallard duck (Anas platyrhynchos) as intermediate host for Sarcocystis wobeseri sp. nov. from the barnacle goose (Branta leucopsis)

“…The 18S rRNA gene investigation results showed that several morphologically similar Sarcocystis species from the cattle and the water buffalo were the same species in both intermediate hosts (Yang et al 2001a;Yang et al 2001b). Molecular studies confirm that S. neurona also use several species as intermediate hosts (Mullaney et al 2005;Mansfield et al 2008;Miller et al 2009). Likewise, in our case, S. wobeseri from the barnacle goose and Sarcocystis sp.…”